Summary: The aims of this research project are to understand the regulation of viral gene expression during herpes simplex virus (HSV) infection, evaluate the feasibility of herpesvirus vectors as gene delivery vectors, and determine the herpesvirus genes that would be most effective for development as potential subunit vaccines. Understanding the regulation of HSV gene expression is essential for the development and evaluation of successful vaccines, particularly live virus vaccines. Similarly, development and evaluation of safe and effective HSV vectors, and the regulated expression of foreign genes in such vectors, requires an understanding of the mechanisms that regulate gene expression. One area of our research is designed to investigate methods which might allow regulated expression of foreign genes in these vectors. In FY02, we have extended our work on replication- incompetent herpes simplex virus vectors that regulate the expression of foreign genes using the tetracycline- response system. These vectors express both a transactivator and a marker gene, each under the control of a tetracycline-response element. Induction of the foreign gene is controlled by the addition of tetracycline analogues in any cell type that can be infected with the vector. Vectors of this type may be especially useful for regulated gene delivery to neurons in vivo and in vitro. We have now expressed neuronal specific genes including that encoding superoxide dismutase in such vectors and are using this system as a tool to study the diffentiated neuron. Another part of this research project is designed to evaluate various HSV gene products as potential components of subunit vaccines, and to evaluate different delivery strategies in order to maximize a protective immune response. Safe and effective vaccines for genital herpes have proven difficult to develop and will likely require a greater understanding of the interaction of the virus with its host as well as the requirements for generation of protective immunity. In FY02, we have evaluated delivery of live HSV vaccines using a bacterial artificial chromosome containing the entire HSV-2 genome. Such a system should facilitate construction of recombinant vaccines by permiting genetic manipulation of the virus in a prokaryotic host. In addition, we have evaluated prime-boost strategies for enhancing the protective immune response to HSV-2 antigens. We have used a plasmid DNA vector that expresses the HSV-2 glycoprotein D gene in combination with a Modified Vaccinia Virus Ankara (MVA) vector expressing the same gene and ashowed that priming with MVA-gD followed by gD-DNA appears to elicit the broadest and most vigorous humoral and cellular response to gD. We are further evaluating this, as well as other, novel vaccination strategies.